High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration–response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration–response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure–activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>105 compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.

https://www.pnas.org/content/pnas/103/31/11473.full.pdf

Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries.

Bronchodilators are central in the treatment of of airways disorders. They are the mainstay of the current management of chronic obstructive pulmonary disease (COPD) and are critical in the symptomatic management of asthma, although controversies around the use of these drugs remain. Bronchodilators work through their direct relaxation effect on airway smooth muscle cells. at present, three major classes of bronchodilators, β(2)-adrenoceptor (AR) agonists, muscarinic receptor antagonists, and xanthines are available and can be used individually or in combination. The use of the inhaled route is currently preferred to minimize systemic effects. Fast- and short-acting agents are best used for rescue of symptoms, whereas long-acting agents are best used for maintenance therapy. It has proven difficult to discover novel classes of bronchodilator drugs, although potential new targets are emerging. Consequently, the logical approach has been to improve the existing bronchodilators, although several novel broncholytic classes are under development. An important step in simplifying asthma and COPD management and improving adherence with prescribed therapy is to reduce the dose frequency to the minimum necessary to maintain disease control. Therefore, the incorporation of once-daily dose administration is an important strategy to improve adherence. Several once-daily β(2)-AR agonists or ultra-long-acting β(2)-AR-agonists (LABAs), such as indacaterol, olodaterol, and vilanterol, are already in the market or under development for the treatment of COPD and asthma, but current recommendations suggest the use of LABAs only in combination with an inhaled corticosteroid. In addition, some new potentially long-acting antimuscarinic agents, such as glycopyrronium bromide (NVA-237), aclidinium bromide, and umeclidinium bromide (GSK573719), are under development, as well as combinations of several classes of long-acting bronchodilator drugs, in an attempt to simplify treatment regimens as much as possible. This review will describe the pharmacology and therapeutics of old, new, and emerging classes of bronchodilator.

Pharmacology and Therapeutics of Bronchodilators

Recent developments in adenosine receptor ligands and their potential as novel drugs

The adenosine A2A receptor is a G-protein-coupled receptor (GPCR) that has been extensively studied during the past few decades because it offers numerous possibilities for therapeutic applications. Herein we describe adenosine A2A receptor distribution, signaling pathways, pharmacology, and molecular structure, followed by a summary and SAR discussion of the most relevant series of adenosine A2A agonists and antagonists. This review also provides an update of the A2A ligands that are undergoing or have undergone clinical studies, including the two currently marketed agonists adenosine and regadenoson.

/pdf/adenosine-a2a-receptor-as-a-drug-discovery-target-2fs6q19dn8.pdf

Adenosine A2A Receptor as a Drug Discovery Target

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins β-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the β(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

/pdf/multiple-ligand-specific-conformations-of-the-b2-adrenergic-irklqh696g.pdf

Multiple ligand-specific conformations of the β2-adrenergic receptor

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled beta(2) adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human beta(2) adrenoceptor (E(max) = 73 +/- 1% of the maximal effect of isoprenaline; pEC(50) = 8.06 +/- 0.02), whereas salmeterol displays only partial efficacy (38 +/- 1%). The functional selectivity profile of indacaterol over beta(1) human adrenoceptors is similar to that of formoterol, whereas its beta(3) adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 +/- 4 min) similar to formoterol and salbutamol, and a long duration of action (529 +/- 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled beta(2) adrenoceptor agonists.

http://jpet.aspetjournals.org/content/jpet/early/2006/01/24/jpet.105.098251.full.pdf

In vitro and in vivo pharmacological characterization of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel inhaled beta(2) adrenoceptor agonist with a 24-h duration of action.

Inhaled β2 adrenoceptor (β2 AR) agonists are widely used as bronchodilator therapies for asthma and COPD. Different agonists have varying rates of onset of action, e.g. indacaterol and salbutamol are effective bronchodilators within 5 min whereas salmeterol takes 15 min to achieve significant bronchodilation over baseline (Brookman et al., Curr Med Res Opin 23:3113–3122, 2007). This has been attributed to differences in the lipophilicity of the agonists such that hydrophobic ligands take longer to diffuse into tissue and may even access the receptor via the membrane compartment (Anderson et al., Eur Respir J 7:569-578, 1994). While this holds true for salmeterol and salbutamol, the relatively high lipophilicity of indacaterol should result in a slower onset of action. Here we have explored the possibility that the efficacy of these ligands may also contribute to their onset of action. We have characterised efficacy and rate of cyclic adenosine monophosphate (cAMP) accumulation in primary human bronchial smooth muscle cells using a competition assay (AlphaScreen, Perkin Elmer) and in HEK 293-GloSensor™ cells endogenously expressing the β2 AR using a luminescence readout. For all agonists tested, cAMP was generated in a concentration-dependent manner. For both assay formats, the relative efficacies were unchanged, with isoprenaline > formoterol > indacaterol > salbutamol > salmeterol. The rate of cAMP generation varied for each agonist and correlated well with intrinsic efficacy in that the high-efficacy agonists promoted the most rapid rise in cAMP levels. We have demonstrated that the rate of cAMP accumulation is influenced by agonist efficacy and that this, in combination with lipophilicity, may explain why β2 AR agonists demonstrate differences in their onset of action.

https://link.springer.com/content/pdf/10.1007%2Fs00210-010-0533-6.pdf

Efficacy is a contributing factor to the clinical onset of bronchodilation of inhaled β2-adrenoceptor agonists

Combinatorial chemistry technology has enabled the synthesis of large, targeted libraries consisting of a much higher ratio of pharmacologically active compounds than traditional compound archives. This has resulted in a higher number of compounds requiring follow-up characterisation in full 50% inhibitory concentration (IC50) curves after the preliminary screen. The aim of this study was to develop a new assay format and analysis protocol for IC50 determination from the minimum number of data points so that more information could be derived from a primary inhibition screen. Data points from existing 10-point IC50 curves were used retrospectively to test the accuracy of IC50 predictions derived from just one or two compound concentrations. Regression analysis showed that both methods were useful, although, as expected, two compound concentrations gave more accurate IC50 predictions. A final experimental data set comparing IC50 values derived from a two- and 10- point assay format gave highly comparable data (r2 = 0.89). This study shows that it is possible to generate IC50 values from an appropriately designed primary inhibition screen using two compound concentrations, reducing the requirement for follow-up IC50 determinations.

Robert J. Turner

Papers

In vitro and in vivo pharmacological characterization of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel inhaled beta(2) adrenoceptor agonist with a 24-h duration of action.

Efficacy is a contributing factor to the clinical onset of bronchodilation of inhaled β2-adrenoceptor agonists

Assessing the minimum number of data points required for accurate IC50 determination.

Synthesis and evaluation of two series of 4'-aza-carbocyclic nucleosides as adenosine A2A receptor agonists.